Disease Models Involving Neovascularization And Squalamine

To determine if squalamine, like the angiostatic steroids previously studied, was effective in disease models involving neovascularization in animals, squalamine was evaluated in two tumor models, the rat 9L glioma allograft and human lung tumor xenografts in mice, and in a neonatal mouse model for corneal neovascularization, which employs hyperoxic exposure (81). The conditions of this model simulate the ocular condition of retinopathy of prematurity (ROP). Although rare, ROP in premature human infants is important, because it can result in blindness.

The rat 9L glioma is widely used as a model for human brain tumors, and for evaluation of the consequences of chemotherapy (82-85). Carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea; BCNU) is an approved agent for the treatment of human brain tumors, and is active in the rat 9L glioma model. BCNU displays cumulative toxicity in animals and humans, so that, when given systemically in animals, a single dose is the common dosage. Systemic squalamine was compared in the rat 9L glioma model to BCNU treatment, when the 9L glioma was transplanted either in the rat brain or in the rat flank. In the latter instance, the blood-brain barrier is not encountered, and drug delivery is less in doubt.

It was found that squalamine given daily by sc or ip injection, beginning on d 3 after transplantation of the 9L glioma, had limited influence on rat survival when the 9L glioma was in the rat brain, but control of 9L glioma growth in the rat flank was similar for BCNU (14 mg/kg, single dose) and squalamine (20 mg/kg daily; Fig. 10). When tumor microvessel density was evaluated by histochemistry with an antibody to the endothelial cell integrin CD34, it was found that squalamine decreased tumor microvessel density in the rat flank model, in concert with tumor volume reduction, but the tumor microvessel density was comparable between control animals and animals treated with BCNU (data not shown). It was also found in this experiment that combined use of BCNU and squalamine was even more effective than either agent alone.

These results suggest that, if the blood-brain barrier is not an issue in drug delivery, squalamine may potentiate the activity of BCNU in restricting the growth of solid tumors, such as the 9L glioma. The degree of tumor control that squalamine has on the 9L glioma in the rat flank was investigated in a subsequent experiment, by varying the frequency of squalamine dosing. It was observed in this study that dosing as infrequently as twice a week considerably reduced the mean and median 9L tumor volume, when scored after 2 wk of treatment, and that tumor volume control improved as the frequency of squalamine dosing was increased (86).

Another study, which further defined possible interaction between squalamine and cytotoxic agents, was conducted with human lung tumor xenografts in nude mice (87). The human lung tumor lines H460 and Calu-6 were implanted subcutaneously in Balb/c athymic nude mice (d 0), and then treated with cisplatin at a maximally tolerated dose (on d 3), squalamine at 10 or 20 mg/kg daily (beginning on d 4), or with cisplatin on d 3 followed with daily squalamine treatment beginning on d 4. Squalamine as a single agent had no significant effect on the growth of either lung tumor line, but squalamine did significantly improve the response of both tumors to cisplatin. This observation remained true for the aggressive H460 tumor growth, even when squalamine was administered only on d 4, suggesting that squalamine has a persistent effect, as was seen with the rat 9L glioma in the rat flank.

Unwanted neovascularization is common to many diseases, of which solid tumors are perhaps the most devastating. Ocular neovascularization is another area of research in which there is interest in limiting angiogenesis to modify the disease outcome. A neonatal mouse model of hyperoxia-induced retinopathy was recently developed by Smith et al. (81), for purposes of understanding the contribution of neovascularization to ultimate eye damage, and for testing the effects of antiangiogenic therapy on the course of the disease. Squalamine was studied in hyperoxygenated mouse neonates, to determine if squalamine could inhibit development of retinopathy without affecting normal retinal vessel growth. Newborn C57Bl/6J mice were exposed to 75% oxygen from postnatal d 7-12, and then allowed to recover in room air. Mice were then given sterile water or 30 mg/kg squalamine subcutaneously daily from d 12-17. Mice were sacrificed at postnatal d 17, and retinal flatmounts were analyzed following fluoroscein-conjugated dextran angiography. It was found that squalamine administration inhibited oxygen-induced retinopathy (p < 0.001), compared to controls (88). Squalamine had no effect on ocular development in room-air-raised control neonates, and also did not have an impact on normal weight gain. These results can be interpreted to mean that squalamine did not have an appreciable effect on normal angiogenesis associated with growth of the animals.